P2X7 receptors (P2X7R) are ATP-gated ion channels expressed in macrophages and dendritic cells (DC). Extracellular ATP, acting via P2X7R, is a highly efficacious stimulus for assembly of inflammasome signaling platforms that drive caspase-1 activation and secretion of biologically active interleukin-12 (IL-12). Inflammasome signaling is involved in an extraordinarily wide array of innate and adaptive immune responses to microbial pathogens or sterile host cell stresses, such as cancer. Recent studies have identified the P2X7R, and its ability to stimulate IL-12 secretion, as critical components of chemotherapy-induced anti-tumor cell immune responses. This response includes: 1) Primary chemotherapy induced-release of ATP from apoptotic tumor cells to activate P2X7R channels in DCs. 2) DC-mediated release of local IL-12 to tumor-reactive T cells that drives their polarization into anti-tumor-effector cells. 3) Lack of this immunogenic component of tumor cell killing in P2X7R-knockout mice which suffer from accelerated tumor growth during chemotherapy. These exciting findings raise several questions. How is P2X7R signaling in DCs initiated during their encounter with apoptotic cells at the tumor locus? What are the mechanisms that initiate and modulate release of ATP from the apoptotic tumor cells? We hypothesize that the efficacy of the P2X7R/IL-12 axis in chemotherapy-induced antitumor immune responses requires that these receptors act as sensors of not only ATP, but other extracellular metabolites, such as NAD and thiol reductants, which accumulate within apoptotic tumor microenvironments via the activation of metabolite-permeable channels. We will use in vitro and in vivo experimental models of murine DC function to test this hypothesis by addressing two major aims: 1) Define the purinergic signaling factors that regulate efficacy of the P2X7R/IL-12 signaling axis in DCs within the tumor microenvironment. These studies will characterize the roles of: a) P2X7R splice variants with distinctive channel gating and agonist selectivities;b) ART2 family ecto-ADP-ribosyltransferases that extracellular NAD to covalently modify and activate P2X7R;c) coactivated G protein-coupled P2Y2 nucleotide receptors or A2b adenosine receptors that may potentiate or attenuate P2X7R signaling and the anti-tumor immunogenic response. 2) Define the mechanisms underlying ATP/NAD release from apoptotic tumor cells and DCs which interact in the anti-tumor immunogenic axis. These studies will: a) quantify extracellular levels of ATP, ATP metabolites, and NAD within apoptotic tumor cell / DC co-cultures;b) characterize the roles of pannexin-1 channels and volume-regulated anion channels as conduits for ATP/NAD release from apoptotic thymocytes, apoptotic tumor cells, and DCs;c) define how ATP-permeable channel activities in apoptotic cells modulate the efficacy of P2X7R/IL-12 signaling within the anti-tumor immunogenic axis. These studies will provide mechanistic insights that may facilitate development of adjuvant purinergic receptor-based therapies which increase the efficacy of immunogenic anti-tumor responses to primary cancer chemotherapeutic agents. PUBLIC HEALTH RELEVANCE: This study proposes to define the cellular signaling mechanisms by which the P2X7 purinergic receptor of inflammatory leukocytes can sense extracellular ATP and other metabolites released from dying tumor cells. Recent studies have identified the P2X7R as an important component of chemotherapy-induced anti-tumor cell immune responses. These studies will provide mechanistic insights that may facilitate development of adjuvant purinergic receptor-based therapies which increase the efficacy of immunogenic anti-tumor responses to primary cancer chemotherapeutic agents.